NOX2 as a biomarker of radiotherapy efficiency in cancer patients

ABSTRACT

Although tumor-associated macrophages have been extensively studied in the control of response to radiotherapy, the molecular mechanisms involved in the ionizing radiation-mediated activation of macrophages remain elusive. Here the present inventors show that ionizing radiation induces the expression of interferon-regulatory factor 5 (IRF5) promoting thus macrophage activation toward a pro-inflammatory phenotype. They reveal that the activation of the Ataxia telangiectasia mutated (ATM) kinase is required for ionizing radiation-elicited macrophage activation, but also for macrophage reprogramming after treatments with γ-interferon, lipopolysaccharide or chemotherapeutic agent (such as cis-platin), underscoring the fact that the kinase ATM plays a central role during macrophage phenotypic switching toward a proinflammatory phenotype. They further demonstrate that NADPH oxidase 2 (NOX2)-dependent ROS production is upstream to ATM activation and is essential during this process. They also report that hypoxic conditions and the inhibition of any component of this signaling pathway (NOX2, ROS and ATM) impairs pro-inflammatory activation of macrophages and predicts a poor tumor response to preoperative radiotherapy in locally advanced rectal cancer. Altogether, these results identify a novel signaling pathway involved in macrophage activation that may enhance effectiveness of radiotherapy through the re-programming of tumor infiltrating macrophages.

SUMMARY OF THE INVENTION

Although tumor-associated macrophages have been extensively studied in the control of response to radiotherapy, the molecular mechanisms involved in the ionizing radiation-mediated activation of macrophages remain elusive. Here the present inventors show that ionizing radiation induces the expression of interferon-regulatory factor 5 (IRF5) promoting thus macrophage activation toward a pro-inflammatory phenotype. They reveal that the activation of the Ataxia telangiectasia mutated (ATM) kinase is required for ionizing radiation-elicited macrophage activation, but also for macrophage reprogramming after treatments with γ-interferon, lipopolysaccharide or chemotherapeutic agent (such as cis-platin), underscoring the fact that the kinase ATM plays a central role during macrophage phenotypic switching toward a proinflammatory phenotype.

They further demonstrate that NADPH oxidase 2 (NOX2)-dependent ROS production is upstream to ATM activation and is essential during this process. They also report that hypoxic conditions and the inhibition of any component of this signaling pathway (NOX2, ROS and ATM) impairs pro-inflammatory activation of macrophages and predicts a poor tumor response to preoperative radiotherapy in locally advanced rectal cancer.

Altogether, these results identify a novel signaling pathway involved in macrophage activation that may enhance effectiveness of radiotherapy through the re-programming of tumor infiltrating macrophages.

BACKGROUND OF THE INVENTION

Radiotherapy is one of the cornerstones of cancer treatment. Approximately, half of all cancer patients are treated with radiotherapy alone or in combination with chemotherapy. Although ionizing radiation (IR) directly causes senescence and death of tumor cells through the generation of reactive oxygen species (ROS) and DNA damage [1], recent studies underscore the fact that IR can also modulate immune cell functions and favor consequently the development of anti-cancer immunity [2, 3]. IR can induce the exposure of “eat-me” signals (such as calreticulin and heat shock protein 70 (HSP70)) and the release of danger signals (such as ATP and high-mobility group box 1 protein (HMGB1)) by the irradiated dying tumor cells (also referred as immunogenic cell death) [4], thus contributing to specific T cell response by increasing the tumor antigen cross-presentation to dendritic cells [5] and/or modifying the immunosuppressive microenvironment of tumors [6, 7]. IR can also control tumor immune response through the direct modulation of innate immune cell functions. Treatment with IR can modulate Langerhans cell functions and induce the accumulation of regulatory T cells into tumors [8]. In addition, IR controls macrophage plasticity and programs tumor-associated macrophages (TAMs) toward pro-inflammatory phenotype that orchestrates specific tumor immune response [9]. However, microenvironment factors such as low oxygen tension (also known as tumor hypoxia) may affect this program and favor the pro-tumorigenic macrophage activation phenotype in response to IR that has been associated with tumor resistance [10]. Nevertheless, the molecular mechanisms underlying IR-induced macrophage activation remain elusive.

Tumor-associated macrophages (TAMs) represent a major cellular component of the tumor microenvironment [11]. These macrophages derive from blood monocytes which, after their recruitment into tumors by tumor-derived chemo-attractants (such as macrophage colony-stimulating factor (M-CSF)), differentiate and activate in response to different environmental signals. Distinct transcriptional programs can be induced to shape their functional phenotypes. Macrophages can be broadly classified as classically activated pro-inflammatory macrophages and alternatively activated pro-tumorigenic macrophages [12]. The transcription factor interferon regulatory factor 5 (IRF5) has been demonstrated to determine the pro-inflammatory macrophage phenotype [13-15] along with other transcription factors (such as signal transducer and activator of transcription 1 (STAT1) and Nuclear factor-κB (NF-κB)), whereas IRF4 [16], STAT6 [17], and Kruppel-like factor 4 (KLF4) [18] are key transcription factors required for the establishment of the pro-tumorigenic macrophage phenotype. Interferon gamma plus lipopolysaccharide (IFN-γ+LPS) or tumor necrosis factor α (TNF-α) alone are known to induce classical macrophage activation that is characterized by an increased secretion of pro-inflammatory cytokines and chemokines (such as interleukin (IL-1β, IL-6, and CXC chemokine ligand (CXCL)8), an augmented production of inducible nitric oxide synthase (iNOS) [19] and of reactive oxygen species (ROS) [12]. Conversely, glucocorticoids, IL-4/IL-13, IL-10 drive macrophages to alternative macrophage activation with a reduced production of pro-inflammatory cytokines but a higher level of anti-inflammatory IL-10, transforming growth factor beta (TGF-β), arginase, membrane scavenger and mannose receptors. Pro-inflammatory macrophages possess bactericidal and antitumoral activities while pro-tumorigenic macrophages are associated with immune regulatory, tissue repair and pro-tumoral activities, highlighting the diversity of macrophage functions. In response to tumor microenvironment signals (such as tumor hypoxia, tumor metabolites and tumor-derived lactate [20, 21]), TAMs mainly adopt a pro-tumorigenic phenotype that contributes to tumor progression by promoting tumor growth, invasiveness and metastasis, angiogenesis and by suppressing immune responses [22-24]. In addition, several studies have demonstrated that TAMs are also associated with treatment resistance and poor clinical outcomes in various cancer settings (such as glioma, lymphoma, melanoma, sarcomas, head and neck, breast and lung cancer [25-28]), making them attractive targets for the development of new anticancer strategies to treat cancer [29, 30]. As the reprogramming of TAMs represents an attractive strategy to improve anti-cancer treatments, a better understanding of the molecular basis of the IR-mediated macrophage activation was needed for the improvement of the efficacy of radiotherapy.

Aware of this need, the present inventors explored the molecular mechanisms involved in IR-induced macrophage reprogramming. They revealed that the subsequent activation of the Ataxia telangiectasia mutated (ATM) kinase controls the expression of the transcription factor IRF5, which is involved in the reprogramming of macrophages towards a proinflammatory phenotype. They found that IR like other classical activators of macrophages increased the expression of the NADPH oxidase 2 (NOX2) which acts upstream of the DNA damage response that is involved in the induction of IRF5 expression and in the pro-inflammatory macrophage activation. They demonstrated that the alteration of this signaling pathway (NOX2→ROS→ATMS1981*) is associated with poor tumor response to neo-adjuvant radiotherapy in locally advanced rectal cancers.

They therefore propose to restore sensitivity and/or responsiveness to anti-cancer treatments (e.g., radiotherapy) by stimulating the activities of the proteins involved in the NOX2/ATM pathway. They also propose to predict the efficiency of an anti-cancer treatment by analyzing in a patient the biomarkers involved in this pathway, in particular ATM and/or NOX2.

DESCRIPTION OF THE INVENTION

Phenotypic and functional plasticity are key features of immune cells (such as macrophages) [38]. Tumor-associated macrophages (TAMs) display a mixed functional phenotype with a majority of alternative features [39] that have been associated with tumor growth and resistance to anti-cancer therapies (such as radiotherapy). Although reprogramming TAMs represents a promising approach to enhance cancer therapies [40-44], molecular mechanisms underlying ionizing radiation-elicited macrophage activation have been poorly characterized.

The present inventors have shown that the in vitro treatment of macrophages with different doses of IR led to their activations toward a pro-inflammatory phenotype. These observations were also confirmed on xenograft tumor models and on human rectal cancer specimens obtained from patients that have been treated with chemoradiotherapy.

Consistent with other in vitro and in vivo studies [13, 15, 45, 46], these results also revealed that the transcription factor IRF5, which is the major regulator of pro-inflammatory macrophage phenotype [15] is activated after IR (as revealed by the up-regulated expressions of IRF5 and IRF5-dependent target genes (such as IL-6, TNF-α or IFN-γ)). The transcription factor IRF5 which has been involved in the expression of pro-inflammatory cytokine genes and in the repression of antiinflammatory cytokine genes (such as IL-10) [15, 47], plays also a central role in hematopoietic cell development [48] and in the susceptibility to inflammatory autoimmune diseases (such as systematic lupus erythematosus, rheumatoid arthritis and multiple sclerosis) [49-51], demonstrating that the expression of IRF5 is tightly regulated during macrophage homoeostasis.

Considering that IR mainly acts through the induction of DNA damages and the production of reactive oxygen species (ROS) [52, 53], the inventors then investigated the role these DNA damage response and ROS production during this process. They report that the DNA damage/repair kinase ATM is activated by IR and by classical macrophage activators (such as LPS and IFN-γ) and that DNA damage/repair ATM kinase is required for macrophage activation. Despite the fact that the ATM kinase may contribute to the development of T cells [54] and of professional antigen-presenting cells (such as macrophage and denditric cells) [55,56] and modulate functions of immune cells (such as STING-dependent macrophage production of type I IFNs [57]), the contribution of ATM to macrophage activation was never investigated. Here, the inventors have found that IR induced the phosphorylations of ATM (on serine 1981) and of the histone H2AX (on serine 139) in a time- and dosedependent manner in macrophages, indicating that DNA double strand breaks and DNA damage response are elicited during macrophage activation. Moreover, the results presented below also demonstrated that ATM inhibition (either by a specific ATM inhibitor or by genetic depletion) impairs IRF5 up-regulation induced by IR and IFN-γ stimulation, highlighting that the biological activity of the kinase ATM controls macrophage activation. Despite several molecular components of DNA damage response pathways (such as Nijmegen breakage syndrome 1 (NBS1) protein [58]) have already been involved in monocyte/macrophage development and functions, the cellular events and signaling pathways that lead to ATM activation and control IRF5 expression during macrophage activation remain to be addressed.

In this study, the inventors also identify the NADPH oxidase 2 (NOX2)-dependent ROS production as an upstream second messenger required for ATM phosphorylation and macrophage activation. They found that the expression of NOX2 was increased after IR or IFN-γ treatment. The NOX2 expression might be regulated at transcriptional level (through the activation of PU.1 or NF-κB transcription factors [35]) or at posttranscriptional level [35]. Increased expression of NOX2 then led to the generation of ROS that contributes to double-strand breaks formation, induces the activation of ATM and IRF5, and favors the functional switch of macrophages from anti-inflammatory to pro-inflammatory phenotype (see FIG. 7 ).

Importantly, higher frequencies of TAMs showing an up-regulation of NOX2 expression (NOX2⁺CD68⁺) were observed in biopsies obtained from “good responders”, as compared to those obtained from “bad responders” (see FIGS. 6F and 6G). This result suggests for the first time that the detection of NOX2 expression on TAMs may serve as a predictive factor for radiotherapy effectiveness.

The present study demonstrates that the histological detection of any components of the molecular cascade described hereafter in macrophages (NOX2→ROS→ATMS1981*→IRF5) predicted the effectiveness of radiotherapy and may also help for the prediction of other anticancer treatments.

In other words, they show for the first time that NOX2, ATM or IRF5 expression on TAMs can be used as a prognostic marker associated with longer overall survival for patients suffering from cancer, notably those that have been (or will be) treated with radiotherapy.

In a first aspect, the present invention relates on a method for in vitro predicting the benefit of the response of a subject diagnosed with cancer to an anti-cancer treatment such as radiotherapy, comprising the steps of:

a) obtaining a biological sample from said subject,

b) determining the NOX2 expression level in said biological sample; and

c) optionally, comparing said NOX2 expression level to the NOX2 expression level of a reference control or population.

In particular, if the NOX2 expression level assessed in step c) for said patient is higher than the reference expression level, then an overall long survival and/or a disease free survival benefit can be predicted. This long survival rate is associated with a low Tumor Regression Grade (typically inferior to 2, according to the Mandard classification). In certain condition, complete or subtotal tumour regression can be predicted.

In a second aspect, the present invention relates on a method for in vitro assessing whether an anti-cancer treatment such as radiotherapy is appropriate for a subject diagnosed with cancer, comprising the steps of:

a) obtaining a biological sample from said subject,

b) determining the NOX2 expression level in said biological sample; and

c) optionally, comparing said NOX2 expression level to the NOX2 expression level of a reference control or population.

In particular, an anti-cancer treatment such as radiotherapy will be determined as an appropriate treatment for said patient if the NOX2 expression level for said patient is higher than the NOX2 expression level for said reference control or population.

Conversely, an anti-cancer treatment such as radiotherapy will be determined as not being appropriate for said patient if the NOX2 expression level for said patient is lower than or equal to the NOX2 expression level for said reference control or population.

In a third aspect, the present invention relates on an in vitro screening method for selecting a subject suffering from cancer for an anti-cancer treatment such as radiotherapy, comprising the steps of:

a) obtaining a biological sample from said subject,

b) determining the NOX2 expression level in said biological sample; and

c) optionally, comparing said NOX2 expression level to the NOX2 expression level of a reference control or population.

wherein said subject will be selected for an anti-cancer treatment such as radiotherapy if the NOX2 expression level for said patient is higher than the NOX2 expression level for said reference control or population.

In these three aspects, the NOX2 expression level can be replaced by the ATM or IRF5 expression level.

As used herein, the expression “biological sample” refers to any sample containing genomic DNA or mRNA from a subject. Said DNA may be contained in a solid tissue, in fluids and/or excretions of said subject. Said fluid is for example blood, serum, plasma, or urine. In a preferred embodiment, said biological sample is a blood sample of said subject, bone marrow or spleen or skin biopsies, or any other cells. Indeed, such a blood sample may be obtained by a completely harmless blood collection from the subject and thus allows for a non-invasive diagnosis. The blood sample used in the method of the invention is preferably depleted of most, if not all erythrocytes, by common red blood cell lysis procedures. The detection is performed on CD68⁺ macrophages.

Macrophages containing samples can be obtained by any convenient means, for example from monocytes containing samples (see example 1.1. below).

Thus, in a more preferred embodiment, the first step of the method of the invention comprises the detection and the measurement of the macrophages present in said biological sample, by conventional markers such as CD11b or CD71.

In an even more preferred embodiment, the method of the invention also comprises the detection and the measurement of the CD68 marker at the cell surface of the macrophages present in the biological sample.

The sequence of the cluster of differentiation CD68 is well-known. The CD68 molecules are glycoproteins that bind to low density lipoproteins. Expression of cell surface CD68 on macrophages may be assessed using specific antibodies, in particular using well known technologies such as cell membrane staining using biotinylation (or other equivalent techniques), followed by immunoprecipitation with specific antibodies, flow cytometry, western blot, ELISA or ELISPOT, antibodies microarrays, or tissue microarrays coupled to immunohistochemistry. Preferably, the expression of cell surface CD68 is detected by flow cytometry. Flow cytometry is a useful tool for simultaneously measuring multiple physical properties of individual particles (such as cells). Cells pass single-file through a laser beam. As each cell passes through the laser beam, the cytometer records how the cell or particle scatters incident laser light and emits fluorescence. Using a flow cytometric analysis protocol, one can perform a simultaneous analysis of surface molecules at the single-cell level. The existence of markers which are specific for each of the contaminant cell types enables the identification of these cells in the blood sample of the subject. Identified contaminant cells can then be removed from the sample (i.e., physically) or from the analysis (i.e., by retaining only the data pertaining to the macrophage population for the analysis), so that the study then only focuses on the macrophage population. In this respect, although any of the above-mentioned analytical techniques can be used to identify the said contaminant white blood cells, flow cytometry is particularly adapted for this task, since it enables the skilled person to eliminate the contaminants and analyze the macrophage population with minimal effort.

Preferably, said biological sample comprises more than 70%, preferably more than 90%, more preferably more than CD68⁺ macrophages.

As used herein, the term “subject” refers to any mammal, preferably a human, suffering from cancer. Yet, the method of the invention is particularly useful for testing a subject suffering from specific cancer, such as a glioma, a lymphoma, a melanoma, a sarcoma, a head and neck tumor, a breast or a lung cancer.

As used herein, a “reference control” corresponds preferably to a control sample comprising cells from “bad responder” patients (which is the reference population as used herein). Bad responders can be characterized by a Tumor Regression Grade superior to 3 (according to Mandard classification as set in [63]). More preferably, said control sample corresponds to CD68⁺ macrophages of said patients. The “control” level of expression of NOX2 therefore corresponds to the average expression of NOX2 in samples collected from “bad responder” patients. Correspondingly, the “control” level of expression of ATM corresponds to the average expression of ATM in samples collected from “bad responder” patients. Finally, the “control” level of expression of IRF5 corresponds to the average expression of IRF5 in samples collected from “bad responder” patients. The expression level of these proteins can be assessed by classical means, as exposed below.

As used herein, the term “anti-cancer treatment” designates any treatment commonly used to treat a patient suffering from cancer. It can be for example a treatment with y-interferon, lipopolysaccharide, a radiotherapy or a chemotherapeutic agent (such as Cis-platin).

As used herein, the term “radiotherapy” or “radiotherapy-based treatment” means a therapy using ionizing radiations. This treatment works by damaging the DNA of cancerous cells. This DNA damage is caused by one of two types of energy, photon or charged particle. This damage is either direct or indirect ionization of the atoms which make up the DNA chain. Indirect ionization happens as a result of the ionization of water, forming free radicals, notably hydroxyl radicals, which then damage the DNA. The amount of radiation used in photon radiation therapy is measured in gray (Gy), and varies depending on the type and stage of cancer being treated. For curative cases, the typical dose for a solid epithelial tumor ranges from 60 to 80 Gy, while lymphomas are treated with 20 to 40 Gy.

As used herein, “NOX2” designates the NADPH oxidase 2 (also known as Cytochrome b-245 heavy chain or cytochrome b(558) subunit beta) which, in humans, is encoded by the CYBB gene. This protein is a super-oxide generating enzyme which forms reactive oxygen species (ROS). The sequence of its mRNA is well-known (NM_000397 as shown in SEQ ID NO:12), as well as the sequence of its protein (NP_000388.2 as shown in SEQ ID NO:13).

In the methods of the invention, the NOX2/ATM/IRF5 expression level can be determined by any means conventionally used by the skilled person, such as PCR, RT-PCR, Northern blot, Western blot, immunohistochemistry, enzyme immunoassay (EIA), radioimmunoassay (RIA), enzyme linked immunoabsorbant assay (ELISA), etc.

In a preferred embodiment, a flow cytometry analysis is performed on the CD68⁺ macrophages isolated from the tested patient (as described above), and the number of NOX2⁺CD68⁺ macrophages is assessed as performed by the inventors in the examples below.

It is known in the art that morphological changes of blood cells begin after 30 minutes of drawing. Such changes consist in granulocyte swelling, increases of band forms, and or loss of specific granulation sometimes associated with vacuolization, especially in eosinophils and monocytes. It will be clear to the skilled person that the results of the method may be affected by the nature and the extent of the changes taking place. It is therefore preferable that the blood sample used in the method of the invention be fresh. By “a fresh blood sample”, it is herein referred to a sample of blood which has been drawn within the previous 48 h, 24 h or 5 hours, preferably 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, or 15 minutes. Preferentially, the fresh blood sample of the invention will be kept at 4° C. until used.

NOX2⁺CD68⁺ macrophages can be detected by using NOX2 antibodies (such as anti-pg91-phox 54.1) and conventional CD68 antibodies, preferably by flow cytometry, as explained in the examples below.

The present invention is also drawn to the use of a kit or array containing a reagent for assaying NOX2 expression, notably for in vitro predicting the benefit of the response of a subject diagnosed with cancer to an anti-cancer treatment such as radiotherapy or for in vitro predicting the benefit of the response of a subject diagnosed with cancer to an anti-cancer treatment such as radiotherapy, or for in vitro selecting a subject suffering from cancer for an anti-cancer treatment such as radiotherapy, in the conditions discussed above.

In a preferred embodiment, this kit contains:

a) a probe and/or or a pair of primers that specifically hybridizes to the NOX2 mRNA or cDNA, or to the complementary sequence thereof; or

b) an anti-NOX2 antibody, optionally labeled, capable of specifically recognizing the NOX2 protein.

In other words, the invention pertains to the use of primers or probes that can specifically amplify or hybridize the genomic region of NOX2, or fragments thereof, or of antibodies which recognize specifically the NOX2 protein (NP_000388.2), for in vitro predicting the benefit of the response of a subject diagnosed with cancer to an anti-cancer treatment such as radiotherapy or for in vitro predicting the benefit of the response of a subject diagnosed with cancer to an anti-cancer treatment such as radiotherapy, or selecting a subject suffering from cancer for an anti-cancer treatment such as radiotherapy.

As used herein, “primers” designate isolated nucleic acid molecules that can specifically hybridize or anneal to 5′ or 3′ regions of a target genomic region (plus and minus strands, respectively, or vice-versa). In general, they are from about 10 to 30 nucleotides in length and anneal at both extremities of a region containing about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers. As they have to be used by pairs, they are often referred to as “primers pair” or “primers set”.

As used herein, “probes” are molecules that are capable of specifically hybridizing a genomic region of interest (e.g., of NOX2mRNA: NM_000397). They are useful to highlight the presence of said genomic region in biological samples. These probes may comprise at least one non-natural nucleotide, e.g., a peptide nucleic acid (PNA), a peptide nucleic acid having a phosphate group (PHONA), a bridged nucleic acid or locked nucleic acid (BNA or LNA), and a morpholino nucleic acid. Non-natural nucleotides also include chemically modified nucleic acids or nucleic acid analogs such as methylphosphonate-type DNA or RNA, phosphorothioate-type DNA or RNA, phosphoramidate-type DNA or RNA, and 2′-O-methyl-type DNA or RNA.

Such analysis can be performed using an antibody (e.g., a radio-labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/ligand pair (e.g., biotin-streptavidin), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which recognize specifically the NOX2 protein of NP_000388.2.

Of note, the inventors demonstrate that the ATM protein, and more specifically the phosphorylated protein form ATMS1981*, may be used alone, or in combination with the NOX2 marker, to predict the efficiency of an anti-cancer treatment, in particular a radiotherapy treatment.

As used herein, “ATM” designates the ataxia telangiectasia mutated protein, which is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks. The sequence of its mRNA is well-known (NM_00051 as shown in SEQ ID NO:15), as well as the sequence of its protein (NP_00042 as shown in SEQ ID NO:14).

Thus, the present invention also relates on a method for in vitro predicting the benefit of the response of a subject diagnosed with cancer to an anti-cancer treatment such as radiotherapy, comprising the steps of:

a) obtaining a biological sample from said subject,

b) determining the ATM expression level in said biological sample; and

c) optionally, comparing said ATM expression level to the ATM expression level of a reference control or population.

In particular, if the ATM expression level assessed in step c) for said patient is higher than the reference expression level, then an overall long survival and/or a disease free survival benefit can be predicted. This long survival rate is associated with a low Tumor Regression Grade (typically inferior to 2 according to the Mandard classification as disclosed in [63]). In certain condition, complete or subtotal tumour regression can be predicted.

Moreover, the present invention relates on a method for in vitro assessing whether an anti-cancer treatment such as radiotherapy is appropriate for a subject diagnosed with cancer, comprising the steps of:

a) obtaining a biological sample from said subject,

b) determining the ATM expression level in said biological sample; and

c) optionally, comparing said ATM expression level to the ATM expression level of a reference control or population.

In particular, an anti-cancer treatment such as radiotherapy will be determined as an appropriate treatment for said patient if the ATM expression level for said patient is higher than the ATM expression level for said reference control or population.

The present invention finally relates on an in vitro screening method for selecting a subject suffering from cancer for an anti-cancer treatment such as radiotherapy, comprising the steps of:

a) obtaining a biological sample from said subject,

b) determining the ATM expression level in said biological sample; and

c) optionally, comparing said ATM expression level to the ATM expression level of a reference control or population.

wherein said subject will be selected for an anti-cancer treatment such as radiotherapy if the ATM expression level for said patient is higher than the ATM expression level for said reference control or population.

In all these methods, the ATM protein is preferably the phosphorylated protein form ATMS1981*. More preferably, this protein is combined with NOX2.

Moreover, the inventors demonstrate that the expression level of the IRF5 transcription factor may be used alone, or in combination with the NOX2 marker and/or the ATM marker, to predict the efficiency of an anti-cancer treatment, in particular a radiotherapy treatment.

As used herein, “IRF5” designates the interferon regulatory factor which acts as a molecular switch that controls whether macrophages will promote or inhibit inflammation. The sequence of its mRNA is well-known (NM_001098627 for isoform b is shown in SEQ ID NO:16; NM_001098629 for isoform d is shown in SEQ ID NO:17), as well as the sequence of its protein (NP_001092097 for isoform b as shown in SEQ ID NO:18; NP_001092099 for isoform d as shown in SEQ ID NO:19).

It is also encompassed a method for in vitro predicting the benefit of the response of a subject diagnosed with cancer to an anti-cancer treatment such as radiotherapy, comprising the steps of:

a) obtaining a biological sample from said subject,

b) determining the expression level in said biological sample; and

c) optionally, comparing said IRF5 expression level to the IRF5 expression level of a reference control or population.

In particular, if the IRF5 expression level assessed in step c) for said patient is higher than the reference expression level, then an overall long survival and/or a disease free survival benefit can be predicted. This long survival rate is associated with a low Tumor Regression Grade (typically inferior to 2 according to the Mandard classification as disclosed in [63]). In certain condition, complete or subtotal tumour regression can be predicted.

Moreover, the present invention discloses a method for in vitro assessing whether an anti-cancer treatment such as radiotherapy is appropriate for a subject diagnosed with cancer, comprising the steps of:

a) obtaining a biological sample from said subject,

b) determining the IRF5 expression level in said biological sample; and

c) optionally, comparing said IRF5 expression level to the IRF5 expression level of a reference control or population.

In particular, an anti-cancer treatment such as radiotherapy will be determined as an appropriate treatment for said patient if the IRF5 expression level for said patient is higher than the IRF5 expression level for said reference control or population.

The present invention also discloses an in vitro screening method for selecting a subject suffering from cancer for an anti-cancer treatment such as radiotherapy, comprising the steps of:

a) obtaining a biological sample from said subject,

b) determining the IRF5 expression level in said biological sample; and

c) optionally, comparing said IRF5 expression level to the IRF5 expression level of a reference control or population.

wherein said subject will be selected for an anti-cancer treatment such as radiotherapy if the IRF5 expression level for said patient is higher than the IRF5 expression level for said reference control or population.

In a preferred embodiment, both the ATM and IRF5 expression levels are combined in the above-methods. In another preferred embodiment, both the NOX2 and IRF5 expression levels are combined in the above-methods.

In a preferred embodiment, the expression levels of all the three proteins (NOX2, ATM and IRF5) is assessed in the above-methods.

Eventually, it has been clearly demonstrated that macrophage activation toward NOX2/ATM dependent pro-inflammatory phenotype was involved in tumor response to chemo-radiotherapy (with no impact on the overall survival and disease free survival of patients).

The present Inventors therefore propose to modulate the components of the NOX2/ATM pathway in order to impact the reprogramming of macrophages elicited by IR or IFN-γ.

This modulation can be an ex vivo activation of the CD68⁺ macrophages that have been extracted from the blood of the patient, and, can be administered to the same patient after their activation. Said activation may be performed by ionizing ex vivo the macrophages of said patients, as disclosed in the examples below (example 2.1.).

Alternatively, this modulation can be performed by stimulating the activity of the NOX2 protein, or of the ATM protein, or of the IRF5 transcription factor. As disclosed in the examples below, stimulating this specific pathway could constitute effective strategies to enhance radiotherapy efficacy in clinic.

Stimulating compounds are for example chosen in the group consisting of:

-   -   NOX2 activators such as small molecules that activate ROS         production, quinolinone derivatives, or NOX agonists with         anti-inflammatory properties that are able to decrease the         pro-inflammatory role of TNF-α in the low nanomolar range [60].     -   ATM activators such as kinases dependent or independent of the         Mre11-Rad50-Nbs1 (MRN) complex, other factors at sites of DNA         breaks, and components that can stimulate ATM         autophosphorylation [61, 62].     -   IRF5 activators such as components that can induce the         phosphorylation of IRF5.

Activation of this pathway can be done by any conventional means, at the transcriptional level, or by activating the activities of the enzyme directly.

The inventors propose that combining radiotherapy with other modalities of cancer treatments (such as PARP inhibitors) might enhance the tumor response to radiotherapy and lead to a long-term benefit to rectal patients.

As used herein, the terms “in vitro” and “ex vivo” are equivalent and refer to studies or experiments that are conducted using biological components (e.g., cells or population of cells) that have been isolated from their usual host organisms (e.g., animals or humans). Such isolated cells can be further purified, cultured or directly analyzed to assess the presence of the target markers. These experiments can be for example reduced to practice in laboratory materials such as tubes, flasks, wells, eppendorfs, etc. In contrast, the term “in vivo” refers to studies that are conducted on whole living organisms.

As used herein, the term “kit” refers to any system for delivering materials. In the context of reaction assays, it includes systems that allow the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials.

FIGURE LEGENDS

FIG. 1 . Irradiation activates macrophages toward pro-inflammatory phenotype.

(A) Colorectal HCT116 cells were injected subcutaneously (4 10⁶ cells/mouse) into immunodeficient mice and tumor growth was monitored. Results are expressed as mean value±SEM. P value (^(δδ)p<0.01) was calculated by means of the paired-Student's t Test. (B-E) Representative confocal micrographs and frequencies of iNOS⁺CD11b⁺ (B, C) or γ-H2AX⁺CD11 b⁺ (D, E) tumor-associated macrophages detected in absence or after 20 Gy single dose irradiation are shown (scale bar, 20 μm). Representative iNOS⁺CD11b⁺ or H2AX⁺CD11 b⁺ macrophages are shown in inserts (scale bar, 5 μm). Results are expressed as mean value±SEM. P value (*p<0.05) was calculated using Mann-Whitney test (n=4). (F-H) Representative confocal micrographs and frequencies of phorbol-12-myristate-13-acetate (PMA)-treated human THP1 monocytes showing γ-H2AX⁺ nuclear foci (F, G) or expressing iNOS (iNOS⁺) (F, H), in control cells or 24 hours after 2 Gy irradiation are shown (scale bar, 20 μm). Representative γ-H2AX⁺ nuclear foci or iNOS expressing macrophages are shown in inserts (scale bar, 5 μm). Results are expressed as mean value±SEM. P values (***p<0.001, ****p<0.0001) were calculated using unpaired Student's t test (n=3). (I-K) IRF5 expression after respectively 96, 96 and 6 hours culture of (PMA)-treated human THP1 monocytes (I), hMDM (J) or murine RAW264.7 macrophages (K) that have been irradiated (or not) with indicated doses. Representative immunoblots are shown (n=3). GAPDH is used as loading control. (L, M) Detection of IL-1β and IL-8 release in the supernatants of hMDM (L) or murine RAW264.7 macrophages (M) that have been irradiated (or not) with indicated doses. Representative immunoblots are shown (n=3). (N, O) Detection of cytokine secretion in the supernatants of hMDMs that have been treated or not with 4Gy irradiation. Array images were captured following 1-10 minute exposures to peroxidase substrate (N). Relative levels of cytokines detected in the supernatants of irradiated macrophages as compared to those detected in non-irradiated macrophages are revealed as fold change of arbitrary units. Pro- and anti-inflammatory cytokines and chemokines are indicated (O). Data are obtained from three healthy representative donors.

FIG. 2 . ATM activation controls IR-induced pro-inflammatory macrophage phenotype.

(A) Representative confocal micrographs of phorbol-12-myristate-13-acetate (PMA)-treated human THP1 monocytes showing γ-H2AX⁺ or 53BP⁺ foci following 2Gy single dose irradiation are shown (scale bar, 20 μm). Scale bar of inserts is 5 μm. (B, C) Frequencies of PMA-treated human THP1 monocytes showing γ-H2AX⁺ (B) or 53BP⁺ (C) nuclear foci after 2 Gy single dose irradiation are shown at indicated times. (D-F) Representative confocal micrographs and frequencies of murine RAW264.7 macrophages showing γ-H2AX⁺ nuclear foci (D, E) or AMTS1981* phosphorylation (ATMS1981*⁺) (D, F), in control cells or 1 hour after 2 Gy single dose irradiation are shown (scale bar, 20 μm). Representative γ-H2AX⁺ nuclear foci and ATMS1981*⁺ macrophages are shown in inserts (scale bar, 5 μm). Results are expressed as mean value±SEM. P values (*p<0.05, **p<0.01,***p<0.001, ****p<0.0001) were calculated using unpaired Student's t Test (n=3). (G, H) ATMS1981*, ATM and IRF5 expression after respectively 96 and 6 hours culture of hMDM (G) or murine RAW264.7 macrophages (H) that have been irradiated (or not) with indicated doses are determined. Representative immunoblots are shown (n=3). Actin is used as loading control. (I-L) Representative confocal micrographs and frequencies of ATMS1981*+CD68⁺ (I, J) or iNOS⁺CD68⁺ (K, L) macrophages that have been detected in absence or after 45 Gy total dose of fractionated irradiation on biopsies obtained from locally advanced rectal cancer patients are shown (scale bar, 20 μm; scale bar of insert, 5 μm). Results are expressed as mean value±SEM. P value (*p<0.05) was calculated using Mann-Whitney test (n=4). (M, N) ATMS1981*, ATM and IRF5 expression after 6 hours culture of murine RAW264.7 macrophages (M, N) that have been depleted for ATM (M) or treated with 20 μM of KU55933 (N) and irradiated (or not) with 2 Gy are shown. Representative immunoblots are shown (n=3). GAPDH (or actin) is used as loading control. (O) ATMS1981*, ATM and IRF5 expression after 96 hours culture of hMDM that have been treated with 10 μM of KU55933 and irradiated (or not) with 4 Gy are shown (n=3). (P) ATMS1981*, ATM and IRF5 expression after 6 hour culture of murine RAW264.7 macrophages that have been treated with 10 μM of Olaparib and irradiated (or not) with 2 Gy are shown. Representative immunoblots are shown (n=3). GAPDH is used as loading control.

FIG. 3 . Classical macrophage activation is dependent of ATM.

(A-C) Representative confocal micrographs and frequencies of murine RAW264.7 macrophages showing γ-H2AX⁺ nuclear foci (A, B) or ATMS1981* phosphorylation (ATMS1981*+) (A, C) in control cells or after 24 h treatments with 20 ng/ml of recombinant murine IFN-γ (mIFN-γ), 100 ng/ml of lipopolysaccharide (LPS) or 10 μM of cisplatinium (CDDP) are shown (scale bar, 20 μm). Representative macrophages with ATMS1981*+ and γ-H2AX⁺ nuclear foci are shown in inserts (scale bar, 5 μm). Results are expressed as mean value±SEM. P values (*p<0.05,**p<0.01,***p<0.001) were calculated using unpaired Student's t Test (n=3). (D-1) ATMS1981*, ATM and IRF5 expressions after 24 hours culture of (PMA)-treated human THP1 monocytes (D, F, G) or murine RAW264.7 macrophages (E, H, 1) that have been treated (or not) with 20 ng/ml of recombinant human IFN-γ (IFN-γ) (D), 20 ng/ml of recombinant murine IFN-γ (m IFN-γ) (E), 100 ng/ml of lipopolysaccharide (LPS) (F), 10 μM of cisplatinium (CDDP) (G, H) or 200 ng/ml of neocarzinostatin (NCZ) (I) are determined. Representative immunoblots are shown (n=3). GAPDH is used as loading control. (J-N) ATMS1981*, ATM and IRF5 expressions after respectively 24 hours culture of murine RAW264.7 macrophages (J, L, M), (PMA)-treated human THP1 monocytes (K) or hMDMs (N) that have been incubated with 10 μM of Olaparib (J), with 20 μM of KU55933 (K, L) or depleted for ATM (M, N) and treated (or not) with 20 ng/ml mIFN-γ (for RAW264.7 macrophages) (J, L, M), 20 ng/ml human IFN-γ (for (PMA)-treated human THP1 monocytes)(K), or 4 μg/ml of human IFN-γ (for hMDM) (N) are evaluated. Representative immunoblots are shown (n=3). Actin (or GAPDH) is used as loading control.

FIG. 4 . Reactive oxygen species are involved in IR-induced pro-inflammatory macrophage activation.

(A-D) Murine RAW264.7 macrophages treated with 1 μg/ml of NAC were stimulated with 2 Gy single dose irradiation (A, B) or 20 ng/ml mIFN-γ (C, D), stained with H2DCFDA and analysed by flow cytometry. Representative flow cytometry analysis and quantifications are shown. Data are presented as means±SEM in B and D panels. Significances are ** P≤0.01,*** P≤0.001 and **** P≤0.0001 (n=3). (E-K) ATMS1981*, ATM and IRF5 expressions after respectively 6 and 24 hours culture of murine RAW264.7 macrophages (E, F, H-K) that have been incubated with 1 μg/ml of NAC (E, F), with cultured in 1% 02 (H, J) or 500 μM DMOG (I, K) and irradiated with 2 Gy single dose (E, H and I) or treated with 20 ng/ml mIFN-γ (F, J and K) were determined. Representative immunoblots are shown (n=3). GAPDH (or Actin) is used as loading control. (G) ATMS1981*, ATM and IRF5 expressions after 48 hours culture of phorbol-12-myristate-13-acetate (PMA)-treated human THP1 monocytes that have been incubated with 10 μM of MnTBAP and irradiated with 8Gy single dose were determined. Representative immunoblots are shown (n=3). GAPDH is used as loading control.

FIG. 5 . NOX2-dependent ROS production is involved in the pro-inflammatory macrophage activation.

(A-E) NOX2 and IRF5 expressions after respectively 96 and 6 hours culture of (PMA)-treated human THP1 monocytes (A) or murine RAW264.7 macrophages (D) that have been irradiated (or not) with indicated doses (A and D); or 24 hours of culture of hMDM with 4 μg/ml of hIFN-γ (B), (PMA)-treated human THP1 monocytes with 20 ng/ml of hIFN-γ(C), or murine RAW264.7 macrophages with 20 ng/ml of mIFN-γ (E), were determined. Representative immunoblots are shown (n=3). GAPDH and actin were used as loading control. (F, G) Representative confocal micrographs and frequencies of NOX2⁺CD68⁺ tumor-associated macrophages detected in absence or after 45 Gy total dose of fractionated irradiation on biopsies obtained from locally advanced rectal cancer patients are shown (scale bar, 20 μm; scale bar of insert, 5 μm). Results are expressed as mean value±SEM. P value (*p<0.05) was calculated using Mann-Whitney test (n=4). (H-K) Murine RAW264.7 macrophages treated with 200 nM of DPI and irradiated with 2 Gy single dose (H, I) or stimulated with 20 ng/ml m IFN-γ (J, K), stained with H2DCFDA and analyzed by flow cytometry. Representative flow cytometry analysis and quantifications are shown. Data are presented as means±SEM in J and L panels. Significances are * P≤0.05, ** P≤0.01, *** P≤0.001 and **** P≤0.0001 (n=3). (L-O) ATMS1981*, ATM and IRF5 expressions after respectively 6 and 24 hours culture of murine RAW264.7 macrophages that have been incubated with 200 nM of DPI (L, M) or depleted for NOX2 (N, O) and irradiated with 2 Gy single dose (L, N) or treated with 20 ng/ml mIFN-γ (M, O) were determined. Representative immunoblots are shown (n=3). GAPDH or Actin was used as loading control.

FIG. 6 . The perturbation of NOX2/ATM-dependent signalling pathway is associated with poor tumor response to radiation therapy.

(A) Densities of CD68⁺ tumour-infiltrating macrophages detected on biopsies of human rectal tumors from good responders (n=29) and bad responders (n=27) to neoadjuvant radiation therapy were analyzed. Data are presented as means±SEM. (B) Representative confocal micrographs and frequencies of ATMS1981*+CD68⁺ (B, C), iNOS⁺CD68⁺ (D, E) or NOX2⁺CD68⁺ (F, G) tumor-associated macrophages detected in absence or after 45 Gy irradiation are shown (scale bar, 20 μm). Representative ATMS1981*+CD68⁺, iNOS⁺CD68⁺ or NOX2⁺CD68⁺ macrophages are shown in inserts (scale bar, 5 μm). Results are expressed as mean value±SEM. P values (** P≤0.01 and *** P≤0.001) were calculated using Mann-Whitney test.

FIG. 7 . Proposed model for the roles of NOX2 and ATM activations in proinflammatory macrophage activation.

EXAMPLES 1. Material and Methods 1.1. Cells and Reagents

The human monocyte cell line THP1 cells and the murine macrophage-like RAW264.7 cells were maintained in RMPI-1640-Glutamax medium (Life technology) supplemented with 10% heat-inactivated fetal bovine serum (Hycultec GmbH) and 100 UI/ml penicillin-streptomycin (Life technology). To obtain THP1 macrophages, THP1 monocytes were differentiated with 320 nM of PMA (Invivogen) during 24 h. Then, cells were washed three times to remove PMA and non-adherent cells. For the generation of monocytes-derived macrophages (hMDM), buffy coats from healthy donor blood were obtained from the French blood bank (Etablissement Français du Sang) under the control of convention with the INSERM. In accordance with French law, written informed consent for the use of cells for clinical research was obtained from each donor. Monocytes were obtained from buffy coats and were differentiated into macrophages by using human AB serum in macrophage medium, as previously described [59]. After seven day differentiation, hMDM were harvested and suspended in macrophage medium containing 10% (vol/vol) heat-inactivated FBS, yielding from 91% to 96% of CD14 positive cells that expressed macrophage differentiation markers (C11b and CD71), and macrophage alternative activation markers (CD163 and CD206). All cells were maintained under 5% CO2 humidified atmosphere at 37° C. For hypoxic experiments, cells were incubated in the hypoxic hood (Scitive) under 1% 02, with 5% CO2 humidified atmosphere at 37° C. Dimethyl Sulfoxide (DMSO), Lipopolysaccharides (LPS), Dimethyloxalylglycine (DMOG), NAcetyl-L-cysteine (NAC), Diphenyleneiodonium chloride (DPI), Neocarzinostatin (NCZ), cis-Diammineplatinum(II) dichloride (CDDP) were purchased from Sigma-Aldrich. Recombinant murine IFN-γ (mIFN-γ) was obtained from PeproTech Recombinant Human IFN-γ was from R&D Systems. KU55933 was from Tocris Bioscience. Mn(III)tetrakis (4-benzoic acid) porphyrin Chloride (MnTBAP) was from Calbiochem. Olaparib was from Selleckchem. Phorbol 12-myristate 13-acetate (PMA) was from Invivogen.

1.2. Antibodies

Antibodies used for immunofluorescence were anti-phospho-ATM (Ser1981), anti-iNOS antibodies from Abcam, anti-53BP1 antibody from Cell Signaling Technology, anti-phospho-H2AX (Ser139) antibody from EMD Millipore. Antibodies used for immunoblots were anti-phospho-ATM (Ser1981) (10H11.E12), anti-ATM (D2E2) antibodies from Cell Signaling Technology; anti-IRF5 and anti-IL-1β antibodies were from Abcam; anti-gp91-phox (54.1) (NOX2) antibody was from Santa Cruz. Anti-GAPDH antibody (EMD Millipore) or anti-beta Actin antibody [AC-15] (HRP) (Abcam) was used as a loading control. For immunohistochemistry staining, antimouse CD11 b (Clone M1/70) antibody was purchased from BD Biosciences; antiphospho-ATM (Ser1981) [EP1890Y] antibody was from GeneTex; anti-gp91-phox (54.1) (NOX2) antibody was from Santa Cruz; anti-phospho-H2AX (Ser139) was from EMD Millipore and anti-iNOS antibody was from Abcam.

1.3. Macrophage Activation

Human MDM (10⁶) were activated by treatment with 2 μg recombinant human IFN-γ for 24 h. THP1 monocytes were differentiated into macrophages by 320 nM PMA for 24 h. Then, macrophages were activated with 20 ng/ml recombinant human IFN-γ or 100 ng/ml LPS during 24 h. RAW264.7 macrophages were activated with 20 ng/ml recombinant murine IFN-γ or 100 ng/ml LPS for 24 h.

1.4. Irradiation

Cells were seeded in 6-well plates, 12-well plates or 25 cm² flasks and irradiated with gamma-ray irradiator IBL-637 (Cs137, 1 Gy/min, gamma CIS-Bio International, IBA, Saclay, France) or with X-ray irradiator (1Gy/min, X-RAD 320, Precision X-Ray). Cells were harvested at indicated time points (hMDMs and THP1 macrophages at 96 h, RAW264.7 macrophages at 6 h) after irradiation for subsequent experiments.

1.5. RNA-Mediated Interference

The SMARTpool siGENOME ATM siRNA (M-003201-04-0005) against ATM, SMARTpool siGENOME CYBB siRNA (M-011021-01-0005) against NOX2 and siGENOME Non-Targeting siRNA Pool #1 (D-001206-13-05) as control were purchased from Dharmacon. siRNA-5 Control, siRNA-4 and siRNA-5 against ATM were from Sigma. Sequences of siRNAs are as follows: SMARTpool siGENOME CYBB siRNAs (containing siRNA-1: 5′ GAA GAC AAC UGG ACA GGA A 3′ (SEQ ID NO: 1); siRNA-2: 5′ GGA ACU GGG CUG UGA AUG A 3′ (SEQ ID NO: 2); siRNA-3: 5′ GUG AAU GCC CGA GUC AAU A 3′ (SEQ ID NO: 3) and siRNA-4: 5′ GAA ACU ACC UAA GAU AGC G 3′ (SEQ ID NO: 4)); SMARTpool siGENOME ATM siRNAs (containing siRNA-1: 5′ GCA AAG CCC UAG UAA CAU A 3′ (SEQ ID NO: 5); siRNA-2: 5′ GGG CAU UAC GGG UGU UGA A 3′ (SEQ ID NO: 6); siRNA-3: 5′ UCG CUU AGC AGG AGG UGU A 3′ (SEQ ID NO: 7); siRNA-4: 5′ UGA UGA AGA GAG ACG GAA U 3′ (SEQ ID NO: 8)); ATM siRNA-5 (5′ UGA AGU CCA UUG CUA AUC A 3′ (SEQ ID NO: 9)); ATM siRNA-6 (5′ AAC AUA CUA CUC AAA GAC A 3′ (SEQ ID NO: 10)) and Control siRNA-5 (5′ UUC AAU AAA UUC UUG AGG U 3′ (SEQ ID NO: 11)). These sequences are listed as SEQ ID NO:1-11 in the enclosed listing. The control siGENOME Non-Targeting siRNAs were a pool of four on-target plus non-targeting siRNAs. INTERFERin™ Reagent (Polyplus Transfection) was used as the siRNA transfection reagent for human monocyte-derived macrophages (hMDM) according to the manufacturer's instructions. Transfection of hMDM was performed as previously described [59].

Briefly, hMDM were seeded (2.5 10⁵ hMDM/0.25 ml/well in 24-well plate in macrophages medium+10% FBS) and were allowed to adhere to the substrate by culturing at 37° C. for 2 hours prior to siRNAs transfection. siRNAs were pre-diluted in 125 μl of Opti-MEM (Thermo Fisher Scientific) in which 10 μl of INTERFERin were then added. The transfection mix was left to incubate at room temperature for 15 minutes and was added to hMDM to achieve the final concentration of 100 nM siRNAs. The MDMs were then incubated at 37° C. for 24 h. The medium was replaced by fresh macrophage medium supplemented with 10% FBS before subsequent experiments. Lipofectamine RNAi max (life technologies) was used to transfect RAW264.7 macrophages according to the manufacturer's instructions. Briefly, RAW264.7 cells were seeded (10⁵ cells/1 ml/well in 12-well plate) and were allowed to adhere to the substrate by culturing at 37° C. for 24 hours prior to siRNAs transfection. The transfection mix was added to the final concentration of 10 nM siRNAs. The RAW264.7 cells were then incubated at 37° C. for 24 h before subsequent experiments.

1.6. Immunofluorescence Microscopy

Cells were grown on coverslips and were treated as indicated. After treatment, cells were rinsed twice, fixed with 10% neutral buffered formalin (Sigma-Aldrich) for 10 min and then permeabilized with 0.3% Triton X-100 in PBS for 15 min. Cells were then washed twice with PBS and were blocked with 10% FBS in PBS for 1 h at room temperature, followed by incubation with primary antibodies (1/100) in 10% FBS in PBS for 1 h30 min at room temperature. Then, samples were incubated with secondary antibodies using Alexa Fluor-488 green or Alexa Fluor-546 red (1/500, Life Technologies) and Hoechst 33342 for nuclei (1/1000, Thermo Fisher Scientific) in 10% FBS in PBS for 30 min at room temperature. Coverslips were mounted with Fluoromount-G (SouthernBiotech) ant then visualized with Leica TCS SPE confocal microscope (Leica Microsystemes, France).

1.7. Immunoblots

Cells were washed twice with cold PBS and lysed with NEHN buffer (0.5% NP40, 20% Glycerol, 300 mM NaCl, 20 mM Herps, pH 7.5, and 1 mM EDTA) complemented with 2.5 mM DTT and the protease and phosphatase inhibitor (Roche) at 4° C. 5-20 μg of proteins were separated by NuPAGE 4-12% or 10% SDS-PAGE gel (Invitrogen) and then were transferred onto a nitrocellulose membrane (0.2 Micron, Bio-Rad). Membranes were blocked with 5% nonfat milk or 5% Bovine Serum Albumine (BSA) in Tris-buffered saline and 0.1% Tween 20 (TBS-T) at room temperature for 1 h and then subsequently probed with primary antibodies (1/5000-1/1000) overnight at 4° C. Then, membranes were incubated with appropriate horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (SouthernBiotech) for 1 h at room temperature. After 3 washes with TBS-T, immunoblots were revealed using G:BOX Chemi XL1.4 Fluorescent & Chemiluminescent Imaging System (Syngene).

1.8. Detection of ROS Production

Hydrogen peroxide and anion superoxide production were determined by staining cells with 5 μM of 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA, Sigma) for 40 min at 37° C. Cells were then washed twice with Hanks' balanced salt solution (HBSS, from Thermo Fisher Scientific) and suspended in cold HBSS solution containing 1% FBS for FACS analysis.

1.9. Determination of LDH Release

The release of LDH in the supernatants of cultured cells was detected using Cytotoxicity Detection KitPLUS (LDH) from Roche according to the manufacturers' instructions.

1.10. Human Cytokine Profiling

Human MDMs were irradiated at 4 Gy and were further incubated for 96 h. The supernatants were harvested, centrifuged and stored at −80° C. until use. Human cytokines in these supernatants were measured using the proteome profiler Human cytokine array panel A (proteome Profiler™) (R&D Systems) according to the manufacturer's instructions. Briefly, membranes were blocked with the blocking buffer at room temperature for 1 h. Supernatants of hMDMs were mixed with a biotinylated detection antibody cocktail and then incubated with the membranes overnight at 4° C. Membranes were washed three times for 10 min and subsequently incubated with streptavidin-horseradish peroxidase for 30 min at room temperature. Membranes were then washed three times for 10 min and exposed to peroxidase substrate and revealed with the G:BOX Chemi XL1.4 Fluorescent and Chemiluminescent Imaging System (Syngene). Time of exposure was between 1 and 10 min. The images were then analyzed using GeneTools software gel image analysis (Syngene).

1.11. In Vivo Mouse Tumor Model

To generate xenograft tumor model, 4 10⁶ human colorectal HCT116 cells were inoculated subcutaneously in the flanks of 5-week female nude mice. Two weeks later, the tumors were irradiated at 20 Gy using Variant-NDI-226-n° 87262-YO X-Ray Tube. Tumor volume was monitored every 4-5 days. Mice were sacrificed when tumors in the control group exceeded 1000 mm³.

1.12. Immunohistochemistry

Tumors obtained from in vivo experiments were resected, fixed and embedded in paraffin. Paraffin-embedded tumor biopsies from rectal patients undergoing neoadjuvant radiotherapy were kindly offered by Dr. Celine Mirjolet in Centre Georges-François Leclerc, Dijon. Frozen tumor biopsies from rectal patients before and after radiotherapy were obtained from Gustave Roussy Cancer Center. Tumor sections were then dried, deparaffinized and hydrated, followed by antigen retrieval with 0.01M Sodium Citrate Buffer, pH 6.0 at 97.6° C. for 20 min. After washing with TBST, slides were blocked with 10% FBS in PBS at room temperature for 1 h. Then primary antibodies diluted in 10% FBS in PBS were applied to each section and incubated overnight in humidified chamber at 4° C. After three washes with TBS-T, Alexa Fluor-conjugated secondary antibodies and Hoechst 33342 diluted in 10% FBS in PBS were applied to each section and incubated for 30 min at room temperature. Then, the slides were washed three times with TBS-T and once with water. Coverslips were mounted on slides using Fluoromount G medium (from SouthernBiotech) before visualization with Leica TCS SPE confocal microscope (Leica Microsystemes, France).

1.13. Human Samples

Human tissue samples of locally advanced rectal tumors that were resected 42 days after receiving 45 Gy (1.8 Gy/sessions) concomitantly to chemotherapy (5-FU) (n=4) or left unirradiated (n=4) were obtained from Gustave Roussy Cancer Campus (Villejuif, France). All tumor samples from responders and non-responders to chemoradiotherapy were obtained from Centre Georges Francois Leclerc (Dijon, France). This study was approved by the IRB and the French CCTIRS committee (Comité consultatif sur le traitement de l'information en matière de recherche et de santé) and CNIL (Commission nationale de l'informatique et des libertés).

Characteristics of the patients are shown on Table 1 below.

Number of patients 56 Age at diagnosis Mean (standard deviation) 65.1 (11.3) Median [min-max] 66.3 [28.7-85.5] Sex Male 36 (64.3%) Female 20 (35.7%) T stage 1-2 5 (8.9%) 3 44 (78.6%) 4 7 (12.5%) N stage 0 21 (37.5%) 1 32 (57.1%) Unknown 3 (5.3%) M stage 0 51 (91.1%) 1 5 (8.9%) TRG 1-2 29 (51.8%) 3-5 27 (48.2%) Number of Gy/fraction Mean (standard deviation) 2.1 (0.5) Median [min-max] 1.8 [1.8-5] <=2 Gy/fraction 43 (76.8%)  >2 Gy/fraction 13 (23.2%) Total doses Mean (standard deviation) 44.1 (3.8) Median [min-max] 45 [25-51] Number of fractionation Mean (standard deviation) 22.9 (4.3) Median [min-max] 25 [5-30] Concomitant Chemotherapy No 25 (44.6%) Yes 30 (53.6%) Unknown 1 (1.8%) Time interval RT surgery Mean (standard deviation) 42.2 (14.2) Median [min-max] 42 [7-71]  <42 days 26 (46.4%) >=42 days 30 (53.6%)

All these patients (n=56) were diagnosed for locally advanced rectal tumors and characterized the Tumor Node Metastasis (TNM) classification. All human samples were obtained after approval by the institutional review board and ethics committee, with fully informed consents.

1.14. Statistical Analysis

All values were presented as mean±SEM for cellular experiments and were analyzed using Student's t-test. Mann-Whitney test was used for results obtained from animal experiments and human biopsies. GraphPad Prism version 6.0b (GraphPad Software) was employed to perform statistical analysis. Multivariate analysis shown in Table 1 was performed using Wald test.

2. Results 2.1. Cell-Autonomous Activation of Macrophages after Ionizing Radiation

Considering that immune cells (such as Th2 cells and regulatory T cells) may influence the functional reprogramming of macrophages [29], IR-mediated macrophage activation were first analyzed using human colon tumor xenografts in immunodeficient mice. Human colorectal HCT116 cells were subcutaneously inoculated into the right flank of athymic nude mice. After seven days, palpable tumors were irradiated with a single dose of 20 Gy, which resulted in significant tumor growth inhibition, as compared to the controls (FIG. 1A). After twenty-nine days, the residual irradiated tumors did not show an increase of the density of CD11b⁺ macrophages (not shown) as compared to the controls, but revealed an increased frequency of CD11b⁺ macrophages that expressed iNOS (iNOS⁺CD11b⁺) (FIG. 1B).

A significant accumulation of iNOS⁺CD11 b⁺ macrophages was detected in irradiated tumors as compared with non-irradiated tumors (FIG. 1C). The accumulation of iNOS⁺CD11b⁺ TAMs positively correlated with tumor response to ionizing radiation, confirming as previously published that the presence iNOS⁺/pro-inflammatory phenotype macrophages in irradiated tumors is required to modify tumor microenvironment and to elicit the tumor regression. Of note, previous reports characterized this process after relatively low dose IR (2 Gy) [6, 9]. TAMs exhibited an increased phosphorylation of the histone variant H2AX on serine 139 (also known as γ-H2AX) (FIGS. 1D and 1E) underlined an unsuspected link between DNA damage response and macrophage activation. To check the possibility whether IR can directly target and activate macrophages, human THP1 macrophages were irradiated with a single dose of 2 Gy and analyzed by fluorescence microscopy, iNOS and γ-H2AX expressions in the irradiated cells. The increase of iNOS and γ-H2AX expressions in irradiated THP1 macrophages (FIGS. 1F-1H) revealed that IR can directly target macrophages to promote their activation toward a pro-inflammatory phenotype. The expression of a central transcription factor involved in macrophage activation, the interferon regulatory factor 5 (IRF5) [15] was then analyzed by immunoblot and it was observed that after single radiation doses of 2 and 4 Gy, phorbol-12-myristate-13-acetate (PMA)-treated human THP1 monocytes (FIG. 1I), human primary monocyte-derived macrophages (hMDM) (FIG. 1J) and murine RAW264.7 macrophages (FIG. 1K) exhibited an enhanced expression of IRF5 (FIGS. 1I-1K). In addition, through immunoprecipitation experiments, it was observed that after 2 Gy irradiation of RAW264.7 macrophages the proinflammatory transcription factor IRF5 was phosphorylated on serine (data not shown). It has also been demonstrated that after respectively 96 and 12 hours of irradiation, hMDM (FIG. 1L) and murine RAW264.7 macrophages (FIG. 1M) released IL-1β and IL-8, two pro-inflammatory cytokines. To further complete the cytokine profile analysis, levels of pro- and anti-inflammatory cytokines were determined and it was observed an increased secretion of pro-inflammatory cytokines (such as IFN-γ, IL-23, IL-6, IL-8 or TNF-α) by 4 Gy-irradiated hMDM as compared with controls (FIGS. 1N and 1O). Altogether, these results indicate that ionizing radiation can promote a cell-autonomous activation of macrophages toward a pro-inflammatory phenotype.

2.2. ATM-Mediated DNA Damage Response Induces IRF5 Expression in Response to Ionizing Radiation

To further characterize molecular mechanisms involved in IR-elicited macrophage activation toward a pro-inflammatory phenotype, the induction of DNA damage-associated signaling pathways was first studied in irradiated macrophages. Fifteen minutes after single radiation dose of 2 Gy, PMA-treated human THP1 monocytes (FIGS. 2A-2C) exhibited a strong nuclear accumulation of γ-H2AX⁺ foci (FIGS. 2A and 2B) and of 53BP1⁺ foci (FIGS. 2A and 2C) that could still be detected 6 hours after irradiation (FIGS. 2B and 2C), revealing that DNA double strand breaks are produced in response to IR. One hour after single radiation dose of 2 Gy, murine RAW264.7 macrophages also displayed increased γ-H2AX⁺ foci (FIGS. 2D and 2E). Considering that the kinase ATM (mutated in the inherited recessive autosomal disease ataxia telangiectasia) is the major kinase involved in the phosphorylation of H2AX (on serine 139) [31], the role of ATM was evaluated in the activation of macrophages in response to IR. The vast majority (more that 80%) of 2 Gy-irradiated murine RAW264.7 macrophages exhibited the activating autophosphorylation of ATM on serine 1981 (ATMS1981*) 1-hour post-irradiation (FIGS. 2D and 2F). These results were confirmed by immunoblots (FIGS. 2G and 2H), which also revealed the positive correlation between ATMS1981* and IRF5 expression when hMDM (FIG. 2G) or murine RAW264.7 macrophages (FIG. 2H) were irradiated with single doses of 2, 4 and 8 Gy. In addition, an increase of ATM phosphorylation/activation in CD68⁺ macrophages was detected in tumor samples after radiotherapy of rectal cancer patients (FIGS. 21 and 2J), as compared to unirradiated patients. The ATM phosphorylation/activation was positively correlated with the increased frequencies of tumor-associated iNOS⁺CD68⁺ macrophages that have been detected 6 weeks after radiotherapy (FIGS. 2K and 2L), demonstrating that the kinase ATM was sustainably activated in macrophages after radiotherapy. The impact of ATM inactivation was next investigated on IR-induced macrophage activation. The depletion of ATM by means of specific small interfering RNA (FIG. 2M) or pharmacological inhibition of ATM with KU55933 (FIGS. 2N and 2O) impaired γ-H2AX and ATMS1981* phosphorylations and the up-regulation of IRF5 expression that was detected respectively, 6 and 96 hours after 2 Gy and 4 Gy single dose irradiation of murine RAW264.7 macrophages (FIGS. 2M, 2N) or hMDM (FIG. 2O) without altering macrophage viability (not shown).

More importantly, an enhancement of ATM activation was also observed through the pharmacological inhibition of poly(ADP-ribose)polymerase (PARP) with Olaparib further enhanced inflammatory macrophage activation elicited by IR (as revealed by the increased expression of IRF5 (FIG. 2P). It was also demonstrated that ATM regulated the expression of IRF5 at transcriptional level (as shown by quantitative real-time (RT) PCR (Data not shown), indicating that the kinase ATM plays a central role during IR-mediated activation of macrophages toward a pro-inflammatory phenotype.

2.3. The Kinase ATM Dictates Classical Macrophage Activation

In order to check whether the activation of ATM was a common feature of pro-inflammatory macrophage activation in response to various agents, the presence of DNA damage-associated nuclear foci was analyzed in response to classical macrophage activators, such as IFN-γ or LPS [32]. Using confocal microscopy, an accumulation of ATMS1981*+ and γ-H2AX+ foci was detected in the nuclei of murine RAW264.7 macrophages that were treated during 24 hours with recombinant murine IFN-γ (mIFN-γ) or LPS (FIGS. 3A-3C). Using immunoblots, it was also observed that the activation of ATM was concomitant with an increased expression of IRF5 in the PMA-treated THP1 monocytes (FIGS. 3D and 3F) or murine RAW264.7 macrophages (FIG. 3E) stimulated with human or murine IFN-γ (FIGS. 3D and 3E) or LPS (FIG. 3F). Interestingly, treatments of these macrophages with some other DNA strand break inducers (such as Cisplatin (FIGS. 3A-3C, 3G and 3H) or neocarzinostatin (FIG. 3I)) or modulators of DNA repair (such as Olaparib (FIG. 3J)), not only activated ATM but also increased IRF5 expression (FIGS. 3G-3J).

The results that are observed in absence of macrophage cytotoxicity suggest that the DNA damage response signaling pathway might be a common pathway involved in classical macrophage activation. Moreover, the pharmacological inhibition (FIGS. 3K and 3L) and the specific depletion (FIGS. 3M and 3N) of ATM inhibited the increase of IRF5 expression that was previously detected after the treatment of PMA-treated THP1 monocytes (FIG. 3K), murine RAW264.7 macrophages (FIGS. 3L and 3M) or hMDM (FIG. 3N) with human or murine IFN-γ (FIGS. 3K-3N), confirming the essential role of the kinase ATM in classical macrophage activation.

2.4. ROS Production Induces ATMS1981* Phosphorylation and IRF5 Expression During Macrophage Activation

Considering that reactive oxygen species (ROS) have been involved in both ATM activation and macrophage differentiation [33, 34], the role of ROS production during macrophage activation was investigated. Using flow cytometry to detect the conversion of the non-fluorescent dye 2,7-dichlorohydro fluorescein diacetate (H2DCFDA) into fluorescent 2,7-dichlorohydro fluorescein (DCF) when ROS are produced, we evaluated the ability of murine RAW264.7 macrophages to generate ROS following IR or mIFN-γ treatment and revealed that both these treatments induced ROS production (FIGS. 4A-4D). Importantly, we demonstrated that the antioxidant N-acetyl cysteine (NAC) and the superoxide dismutase (SOD) mimetic Mn(III)tetrakis (4-benzoic acid) (MnTBAP) that blunted the ROS production (FIGS. 4A-4D), inhibited also the activating phosphorylation of ATM (ATMS1981*) (FIGS. 4E-4G) and reduced the increased expression of IRF5 (FIGS. 4E-4G) that we observed after treatment with IR (FIGS. 4E and 4G) or mIFN-γ (FIG. 4F) of RAW264.7 macrophages (FIGS. 4E and 4F) or treatment with IR of PMA-treated THP1 monocytes (FIG. 4G) without impacting macrophage viability (not shown). These results suggested that ROS production may dictate the classical pro-inflammatory state of macrophages.

Then, it was assessed whether low oxygen tension (also known as hypoxia) which is a physio-pathological situation known to reduce the generation of ROS [35] may also impact the activation of macrophages. The effects of hypoxic conditions (1% oxygen) and of a small molecule inhibitor of prolyl hydroxylase domain (PHD)-containing proteins, the dimethyloxallyl glycine (DMOG) (which mimics hypoxia) were thus evaluated on IR-mediated pro-inflammatory macrophage activation. It was observed that IR-mediated ATMS1981* and IRF5 expressions were reduced in RAW264.7 macrophages that have been incubated in hypoxic conditions (FIG. 4H) or treated with DMOG (FIG. 4I), as compared to control cells. The results were confirmed after stimulation of murine RAW264.7 macrophages with mIFN-γ (FIGS. 4J and 4K). Altogether, these results indicate that, by controlling activating phosphorylation of ATM and the induction of the IRF5, ROS produced in the stimulated macrophages play a key role in the activation process.

2.5. The NADPH Oxydase (NOX2) is Responsible for ROS Production and ATM Phosphorylation During Macrophage Activation

The NADPH oxidases (NOX) and dual oxidase (DUOX) are major regulated sources of ROS generation [36]. To characterize mechanisms that are involved in ROS generation during macrophage activation, the role of NADPH oxidase 2 (NOX2), which is mainly expressed in macrophages and neutrophils [35, 36], was examined. First, using immunoblots, it was observed that NOX2 was up-regulated after irradiation of PMA-treated THP1 monocytes (FIG. 5A) with single doses of 2 and 4 Gy. These results were confirmed with the treatments of hMDM (FIG. 5B), PMA-treated THP1 monocytes (FIG. 5C) and RAW264.7 macrophages (FIGS. 5D and 5E) with human or murine IFN-γ (FIGS. 5B, 5C and 5E) or IR (FIG. 5D). NOX2 expression was also found increased in CD68⁺ macrophages that were detected in tumor samples obtained 6 weeks after radiotherapy of rectal cancer patients (FIGS. 5F and 5G), as compared to biopsies obtained from the same patients before radiotherapy. To precise the role of NOX2 during macrophage activation, the effect of the pharmacological NADPH oxidase inhibitor, diphenylene iodonium (DPI) was evaluated on ROS production, ATMPS1981* and IRF5 up-regulation detected after the treatment of RAW264.7 macrophages with IR (FIGS. 5H, 5I and 5L) or mIFN-γ (FIGS. 5J, 5K and 5M). It was demonstrated that DPI impaired all events of the above-described signaling cascade (FIGS. 5H-5M) without modifying macrophage viability (not shown). In addition, depletion of NOX2 with specific small interfering RNA on irradiated (FIG. 5N) or mIFN-γ treated (FIG. 5O) RAW264.7 macrophages also reduced ATMS1981* and IRF5 up-regulation as revealed by immunoblots, in comparison to control cells. These results demonstrate that the induction of NOX2-dependent ATM activation is required for tuning macrophages toward a pro-inflammatory phenotype.

2.6. The Alteration of NOX2/ATM-Dependent Tumor Macrophage Activation is Associated with Poor Prognosis after Radiotherapy

Despite the fact that neo-adjuvant chemo-radiotherapy for locally advanced rectal cancer patients improved local control of the tumors, only 15% of patients exhibit a complete response to treatment [37]. In this context, it was analyzed whether the perturbation of the signaling pathway (NOX2→ROS→ATMS1981*) involved in the macrophage activation toward a pro-inflammatory phenotype may be associated with the absence of local response to radiotherapy. Resected specimens of rectal cancer patients obtained after neo-adjuvant radiotherapy that have been performed before radical tumor resection were analyzed. According to the tumor regression grade (TRG) criteria of Mandard et al ([63]), these patients were classified into “good responders” (TRG≤2, n=29) and “bad responders” (TRG≥3, n=27) (Table 1 above). The total number of CD68⁺ tumor-associated macrophages were analyzed in both groups of irradiated tumors and significant difference in the CD68⁺ TAMs infiltration was not detected (FIG. 6A). In addition, the auto-phosphorylation ATMS1981* was detected in approximately 20% of TAMs (FIG. 6B), but a significant difference on the frequencies of TAMs exhibiting the auto-phosphorylation ATMS1981* (ATMS1981*+CD68⁺) was not observed between the both groups of tumors (FIG. 6C).

Interestingly, it was detected a significant increase in the frequency of TAMs revealing an enhanced expression of the inducible nitric oxide synthase (iNOS⁺CD68⁺) on tumor samples obtained from “good responders” as compared to those obtained from “bad responders” (FIGS. 6D and 6E), confirming that macrophage activation toward a pro-inflammatory phenotype is associated with the local tumor control.

Finally, higher frequencies of TAMs showing an up-regulation of NOX2 expression (NOX2⁺CD68⁺) were observed in biopsies obtained from “good responders”, as compared to those obtained from “bad responders” (FIGS. 6F and 6G), revealing that the detection of NOX2 expression on TAMs may serve as a predictive factor for radiotherapy effectiveness. Multivariate statistical analysis confirmed these results (Table 2).

TABLE 2 Multivariate analysis of macrophage histological markers in rectal cancer response to neoadjuvant radiotherapy. The statistical comparisons of indicated histological markers between “good responders” (TRG ≤ 2, n = 29) and “bad responders” (TRG ≥ 3, n = 27) have been adjusted on TNM stages, time interval between radiotherapy and surgery and concomitance with chemotherapy. Median cuts off, Odds Ratios (OR) and 95% confidence interval (95% CI) are indicated. P values were calculated using Wald test. OR TRG (3-4-5) vs (1-2) 95% CI P-value CD68+/mm2 0.635 median cut off  <376.23 1 >=376.23 1.369 [0.374-5.010] iNOS+/CD68+ (%) 0.003 median cut off  <53.72 1  >=53.72 0.089 [0.018-0.431] NOX2+/CD68+ (%) 0.006 median cut off  <55.07 1  >=55.07 0.077 [0.013-0.472] ATMS1981*+/CD68+ (%) 0.339 median cut off  <14.93 1  >=14.93 0.513 [0.131-2.013]

Altogether, these results confirm that the NOX2→ROS→ATMS1981* cascade may contribute to an efficient macrophage activation in response to radiotherapy.

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The invention claimed is:
 1. A method for treating cancer, comprising the steps of: a) selecting a tumor sample from a subject diagnosed with cancer that comprises CD68+ macrophages, b) measuring by flow cytometry the NOX2 expression level of the CD68+ macrophages; c) measuring the NOX2 expression level of a reference sample comprising CD68+ macrophages from radiotherapy bad responder patients characterized by a Tumor Regression Grade superior to 3 after radiotherapy, and d) administering radiotherapy to the subject, wherein the subject has a NOX2 expression level in CD68+ macrophages higher than in said reference sample NOX2 expression level.
 2. The method according to claim 1, wherein the selected sample and the reference sample comprise more than 70% CD68+ macrophages.
 3. The method of claim 1, wherein said subject is a human patient.
 4. The method of claim 1, wherein said subject suffers from a glioma, a lymphoma, a melanoma, a sarcoma, a head and neck tumor, a breast cancer, or a lung cancer.
 5. The method of claim 1, wherein the NOX2 expression level in the selected sample and the reference sample is determined using an anti-NOX2 antibody labeled for flow cytometry.
 6. The method of claim 1, wherein the tumor sample is a solid tumor tissue. 